Vitellogenin is used as a biomarker for estrogenic effect in laboratory-based regulatory test systems OECD TGs 229, 230 and 234 and vitellogenin levels in the plasma of juvenile or male specimens of various fish species have also been used for monitoring estrogenic impacts in freshwater and estuarine environments, especially in association with discharges from waste water treatment plants. The use of any biomarker requires knowledge about the baseline levels in an uncontaminated environment and the present investigation was initiated to identify baseline vitellogenin levels in juvenile brown trout Salmo trutta. Juvenile brown trout were caught by electro-fishing from 15 sites in 12 streams of Funen, Denmark, during late September and early October 2010. The fish were brought to the laboratory, blood samples were taken and plasma vitellogenin concentrations were determined by means of a direct non-competitive sandwich ELISA. Vitellogenin concentrations in the plasma of the 135 male, juvenile brown trout caught during the investigation were all below 52 ng/ml and vitellogenin concentrations in the juvenile, female brown trout were generally one order of magnitude higher. The results indicate that baseline vitellogenin levels in juvenile, male brown trout in an uncontaminated environment are below 100 ng/ml. This also indicates that most of the high values obtained in earlier investigations (2000 to 2004) investigations could be attributed to estrogenic exposure. Six of the streams investigated in 2004 were revisited in the 2010 study and vitellogenin concentrations had decreased. Between 2004 and 2010 some discharges (via septic tanks) from scattered houses in the open land known to discharge estrogenic activity had been removed and this may have reduced the addition of estrogenicity to the streams.